Construction and characterization of chimeric enzymes of kojibiose phosphorylase and trehalose phosphorylase from Thermoanaerobacter brockii

Chimeric phosphorylases were constructed of the kojibiose phosphorylase (KP) gene and the trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii. Four chimeric enzymes had KP activity, and another had TP activity. Chimera V-III showed not TP, but KP activity, although only 125 amino acid residues in 785 residues of chimera V-III were from that of KP. Chimera V-III had 1% of the specific activity of the wild-type KP. Furthermore, the temperature profile and kinetic parameters of chimera V-III were remarkably changed as compared to those of the wild-type KP. The results of the molecular mass of chimera V-III using GPC (76,000 Da) strongly suggested that the chimera V-III protein exists as a monomer in solution, whereas wild-type KP and TP are hexamer and dimer structures, respectively. The result of the substrate specificity for phosphorolysis was that the chimera acted on nigerose, sophorose and laminaribiose, in addition to kojibiose. Furthermore, chimera V-III was also able to act on sophorose and laminaribiose in the absence of inorganic phosphate, and produced two trisaccharides, beta-D-glucosyl-(1-->6)-laminaribiose and laminaritriose, from laminaribiose.     

H2BC: a new technique for NMR analysis of complex carbohydrates

It is demonstrated that the H2BC NMR pulse sequence (J. Am. Chem. Soc.2005, 127, 6154, Magn. Reson. Chem.2005, 43, 971-974) offers unambiguous assignments and significant simplification of NMR spectra of large and complex carbohydrates compared to other techniques for the establishment of correlations over more than one bond. H2BC almost exclusively correlates protons and proton-bearing carbon spins separated by two covalent bonds and is independent of occasionally vanishing (2)J(CH) coupling constants, which alleviates the problem of missing two-bond correlations in HMBC spectra. H2BC also solves the problem of distinguishing two- and three-bond correlations in HSQC-TOCSY or HMBC. It is a further asset of H2BC that the experiment is significantly shorter than HMBC and HSQC-TOCSY, and hence less sensitive to transverse relaxation. The H2BC experiment is demonstrated on an approximately 30-residue oligosaccharide from Francisella victoria.     

Synthesis of a novel N[bond]O-interglycosidic disaccharide

The carbohydrate subunits carrying an N-O-interglycosidic bond play a very important role in the biological activity of the enediyne antibiotics. Condensation of O-(alpha- and beta-D-glucopyranosyl)hydroxylamine (5a and 5b) with the hex-3-ulopyranoside (6) furnished methyl 4,6-O-benzylidene-2,3-dideoxy-3-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyloxy)imino-alpha- and beta-D-erythro-hexopyranoside (7a and 7b). Stereoselective reduction of the Cz.dbnd6;N bond of 7a and 7b with sodium cyanoborohydride resulted in the formation of the required protected N-O-interglycosidic disaccharides (8a and 8b). Finally, catalytic hydrogenation of 8a afforded methyl 2,3-dideoxy-3-(alpha-D-glucopyranosyloxy)amino-alpha-D-ribo-hexopyranoside (9a). Under similar conditions the beta anomer 8b underwent decomposition.     

Conformational studies of a novel cationic glycolipid,glyceroplasmalopsychosine, from bovine brain by NMR spectroscopy

A novel glycosphingolipid containing a long chain aldehyde conjugated to galactose and glycerol, Gro1(3)-O-CH((CH(2))(n)CH(3))-O-6Galbeta-sphingosine (glyceroplasmalopsychosine) has been studied by NMR spectroscopy (Hikita et al. J. Biol. Chem. 2001, 276, 23084-23091). We further report here on the conformation showing the galactose and the glycerol at the end of two parallel hydrophobic chains, i.e. the sphingosine and the fatty aldehyde. This is proposed based on the interproton distances derived from ROESY experiments and 3 J (H,H) coupling constants. The absence of any intraresidual NOEs between protons in the glycerol residue suggested that the C-C-2 and C-C-3 bonds in the glycerol may be rotating freely, supporting the proposed conformation in which the unique terminal glycerol is in an environment with a minimal steric hindrance. The present study proposes a conformation of glyceroplasmalopsychosine greatly different from the two conventional plasmalopsychosines possessing a fatty aldehyde chain oriented in an opposite direction to the sphingosine.     

Synthesis of aryl sialosides using Mitsunobu conditions

Mitsunobu conditions for the efficient synthesis of aryl alpha/beta-sialosides were developed. An oxidative work-up procedure was employed to avoid a cumbersome chromatographic separation from the 2,3-dehydro derivative of sialic acid, which is formed as a side-product.     

Synthesis and solid state 13C and 1H NMR analysis of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and ester of amino acids or dipeptides

The syntheses of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and amino acid or peptide esters are presented. The reaction of methyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside and oxalyl chloride gave N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl) oxamic acid chloride which on reaction with the ester of Gly, L-Ala, L-Phe, GlyGly, Gly-L-Phe and Gly-L-Ala afforded N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl), N'-oxalyl-amino acid or dipeptide esters. The structure of the oxamides was studied using 1H, 13C NMR in solution and solid state.     

Determination of the three-dimensional structure of oligosaccharides in the solid state from experimental 13C NMR data and ab initio chemical shift surfaces

A novel method for the determination of the three-dimensional (3D) structure of oligosaccharides in the solid state using experimental 13C NMR data is presented. The approach employs this information, combined with 13C chemical shift surfaces (CSSs) for the glycosidic bond carbons in the generation of NMR pseudopotential energy functions suitable for use as constraints in molecular modeling simulations. Application of the method to trehalose, cellobiose, and cellotetraose produces 3D models that agree remarkably well with the reported X-ray structures, with phi and psi dihedral angles that are within 10 degrees from the ones observed in the crystals. The usefulness of the approach is further demonstrated in the determination of the 3D structure of the cellohexaose, an hexasaccharide for which no X-ray data has been reported, as well as in the generation of accurate structural models for cellulose II and amylose V6.     

Mononuclear Au(III)-complexes with tryptophan-containing dipeptides: Synthesis, spectroscopic and structural elucidation

The coordination ability of dipeptides l-tryptophyl-l-phenylalanine (H-Trp-Phe-OH) and l-tyrosyl-l-tryptophan (H-Tyr-Trp-OH) with Au(III) have been elucidated both in solid state and in solution by means of series of methods as UV, ¹H and ¹³C NMR, conventional and linear-polarized IR-spectroscopic tool in solid-state, based on orientation technique as suspension in nematic liquid crystal, FAB-MS, TGV, DSC methods and elemental analysis. The structures of the Au(III)-complexes have been predicted theoretically by DFT calculations at B3LYP level of theory and Lanl2DZ (Au)/6-31+G(3df) (Cl, C, H) basis set. The last data are compared with IR-LD spectroscopic ones giving the experimental evidence of the structures of the complexes studied. The dipeptides interact as tridentate ligands in obtained mononuclear complexes via their -NH₂, deprotonated N⁻-amide and -groups at molar ratio metal to ligand 1:1. One Cl⁻ ion is joined to the Au(III) as terminal ligand, forming [Au^III(LH₋₁)Cl] species. A near to square-planar flat geometry of the chromophores AuN₂OCl is yielded with maximal deviation of total planarity less than 0.9°.     

Synthesis, crystal structure and DNA interaction studies on mononuclear zinc complexes

A new family of mononuclear Zn(II) complexes [Zn(Pyimpy)₂](ClO₄)₂ (1), [Zn(Pyimpy)(Cl)₂] (2), [Zn(Pyimpy)(SCN)₂] (3) and [Zn(Pyimpy)(N₃)₂] (4) were synthesized using designed tridentate ligand Pyimpy having NNN donors (Pyimpy: (2-((2-phenyl-2-(pyridin-2-l)hydazono)methyl)pyridine)). Complexes were characterized by different spectroscopic studies and it has been found out that all complexes exhibited strong fluorescent emission at room temperature. Molecular structures of [Zn(Pyimpy)₂](ClO₄)₂·C₆H₅CH₃·0.5H₂O (1·C₆H₅CH₃·0.5H₂O) and [Zn(Pyimpy)(Cl)₂]·CH₃CN (2·CH₃CN) were determined by X-ray crystallography and ligand coordinated Zn(II) ions was described as distorted octahedral and distorted square pyramidal, respectively. DNA binding properties of these complexes were investigated by absorption spectral, fluorescence quenching and circular dichroism spectral studies.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

NMR and modelling studies of disaccharide conformation

Long-range heteronuclear coupling constants were measured across the glycosidic linkages for a series of eight alpha- or beta-linked disaccharides in aqueous solution. Multiple 13C site-selective excitation experiments using 1H decoupling in conjunction with pulsed field gradient-enhanced spectroscopy were used to determine 3J(C,H) values. These were subsequently compared with the respective couplings calculated, using a Karplus relationship, from molecular dynamics simulations with the explicit inclusion of water.     

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

Summary The assignments of the ¹H, ¹⁵N, ¹³CO and ¹³C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled ¹⁵N- and ¹⁵N-/¹³C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic ¹H and ¹³C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as ³J_H n _H coupling constants, amide exchange and ¹³C and ¹³C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.     

1,5-Anhydroxylitol from leaves of Olea europaea

1,5-Anhydroxylitol, a compound never found previously in the vegetal kingdom was obtained from Olea europaea leaves in approximately 0.5-1% yield.     

Metabolism of [1-13C]glucose in a synaptosomally enriched fraction of rat cerebrum studied by1H/13C magnetic resonance spectroscopy

This study explored the utility of¹H and¹³C magnetic resonance spectroscopy to study a standard synaptosomally enriched fraction (P2 pellet) made from rat cerebrum. The preparations contained high concentrations of N-acetylaspartate and -aminobutyric acid and low concentrations of glutamine, indicating that they were in fact rich in neuronal cytosol. The metabolic competence of the preparation was assessed by quantitative measurements of its ability to convert [1-¹³C]glucose into lactate, glutamate, aspartate, and other metabolites under well oxygenated conditions in 30 minutes. The minimum mean glycolytic rate was 0.8 mM glucose/min and the flow through the tricarboxylic acid cycle was equivalent to 0.2 mM glucose/min.Abbreviations ppm parts per million (chemical shift scale) - NMR nuclear magnetic resonance - GABA -aminobutyric acid - PBS phosphate-buffered normal saline solution - TSP 3-trimethylsilylpropionate During the performance of these studies Dr. A.P. Burlina was on leave from Instituto di Clinica delle Malattie Nervose e Mentali, University of Padua, Padua, Italy.     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

Imazalil-cyclomaltoheptaose (beta-cyclodextrin) inclusion complex: preparation by supercritical carbon dioxide and 13C CPMAS and 1H NMR characterization

An inclusion complex between imazalil (IMZ), a selected fungicide, and cyclomaltoheptaose (beta-cyclodextrin, betaCD) was obtained using supercritical fluid carbon dioxide. The best preparation conditions were determined, and the inclusion complex was investigated by means of 1H NMR spectroscopy in aqueous solution and 13C CPMAS NMR spectroscopy in the solid state. Information on the geometry of the betaCD/IMZ complex was obtained from ROESY spectroscopy, while the dynamics of the inclusion complex in the kilohertz range was obtained from the proton spin-lattice relaxation times in the rotating frame, T(1rho) (1H).     

1,3-Dideoxynojirimycin-3-yl glycosides of beta-(1-->3)- and beta-(1-->6)-linked gluco-oligosaccharides

Standard chemical methods involving the use of O-acetylated glycosyl trichloroacetimidates as glycosylating agents were used to prepare the five 1,3-dideoxynojirimycin-3-yl beta-(1-->3)-linked oligo-glucosides (1-5) and also the beta-(1-->6)-bonded glucobiose (gentiobiose)-based analogue 6 as potential fungicides. In the course of the work, the beta-(1-->6), beta-(1-->6)-linked analogue 8 of 6 and 6-O- and 4-O-beta-glucopyranosyl-deoxynojirimycins 7 and 9, respectively, were also produced.     

Assigning stereodiversity of the 27-Me group of furostane-type steroidal saponins via NMR chemical shifts

Applicability of (13)C and (1)H NMR chemical shifts for the assignment of the 25R/25S configuration of the 27-methyl group in the case of furostane-type steroidal saponins has been investigated. A comparative study of (13)C NMR data suggest that chemical shift values for C-20, C-21, C-22, C-23, C-24, C-25, C-26 and C-27 resonances were not much influenced by R/S configuration of the 27-Me group, thus reflecting limited application of (13)C NMR chemical shifts for such stereochemical determinations. In contrast, (1)H NMR chemical shifts (delta(a), delta(b)) for geminal protons of glycosyloxy methylene (H(2)-26) exhibit pronounced dependence and the difference (Delta(ab)=delta(a)-delta(b)) among their chemical shifts [Delta(ab)= or <0.48 for 25R; Delta(ab)= or >0.57 for 25S] seems to be of general applicability for ascertaining 25R/25S orientation of the 27-methyl group of furostane-type steroidal saponins.